Microscopy imaging device with advanced imaging properties

ABSTRACT

Systems, methods and devices are implemented for microscope imaging solutions. One embodiment of the present disclosure is directed toward an epifluorescence microscope. The microscope includes an image capture circuit including an array of optical sensor. An optical arrangement is configured to direct excitation light of less than about 1 mW to a target object in a field of view of that is at least 0.5 mm 2  and to direct epi-fluorescence emission caused by the excitation light to the array of optical sensors. The optical arrangement and array of optical sensors are each sufficiently close to the target object to provide at least 2.5 μm resolution for an image of the field of view.

FEDERALLY-SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under contractDE-A52-07NA27344 awarded by the Department of Energy. The Government hascertain rights in this invention.

OVERVIEW OF CERTAIN EMBODIMENTS

Aspects of the present disclosure relate generally to microscopy imagingdevices, for example, miniature epifluorescence imaging devices.

Optical microscopes are often designed as instruments of substantialsize and expense. The role of imaging in biomedicine has grown, andminiaturized integration of the light microscope facilitates theadvancement of many new applications. For instance, mass-producible,tiny microscopes, can be useful for imaging of cells in freely behavinganimals, and particularly in the brain, for which is useful forunderstanding how cellular dynamics relate to animal behavior.

Although not limited thereto, aspects of the present disclosure relateto miniature (<2 g), integrated fluorescence microscopes made frommass-producible parts, including a semiconductor light source and imagesensor, allowing imaging across ˜0.5 mm² areas. Such devices can beconfigured for high-speed observation of cellular dynamics withsufficient image quality and/or resolution that such observation isuseful for viewing dynamics of the brains of active mice at frameacquisition rates up to 100 Hz. The use of a miniature microscope can beuseful for a variety of different applications (e.g., trackingCa2+-spiking concurrently in up to >200 Purkinje neurons extending over9 cerebellar microzones).

Aspects of the present disclosure are directed toward epifluorescencemicroscopes. The microscope includes an image capture circuit with anarray of optical sensors. An optical arrangement is configured to directexcitation light of less than about 1 mW to a target object in a fieldof view that is at least 0.5 mm² and to direct epi-fluorescence emissioncaused by the excitation light to the array of optical sensors. Theoptical arrangement and array of optical sensors are each sufficientlyclose to the target object to provide at least 2.5 μm resolution for animage of the field of view.

Certain embodiments of the present disclosure are directed to anepifluorescence microscope that has an optical light source configuredto produce excitation light from an energy source that provides lessthan 6 mW. The microscope includes an imaging circuit including a sensorarray and an objective lens configured to operate sufficiently close tothe optical light source, the image sensor array and to a target objectto provide at least 2.5 μm image resolution for the field of view thatis at least 0.5 mm².

Other embodiments of the present disclosure relate to epifluorescencemicroscopes that occupy less than a cubic inch. Such a microscopeincludes an optical excitation arrangement configured to direct lighttoward a field of view containing an imaging target. An imaging circuitincluding optical sensor array is configured to generate image data fromfluorescence caused by an interaction between the directed light and theimaging target. An optical arrangement is configured to direct thefluorescence to the optical sensor array with sufficient intensity andfocus for the image data to depict over 0.20 mm² and a resolution of atleast 3 μm. In other embodiments the intensity and focus for the imagedata is sufficient to depict at least 2.5 μm image resolution for thefield of view that is at least 0.5 mm²

Consistent with other embodiments of the present disclosure, an imagingdevice includes a portable housing that is less than a cubic inch insize. The portable housing contains several elements including anexcitation source configured to provide excitation light. A structure isalso included, the structure being configured to provide an opticalpathway having a first end and a second end. The structure includes anobjective lens at the first end of the optical pathway; one or moreexcitation elements that are configured and arranged to direct theexcitation light to the objective lens; and one or more emissionelements that are configured and arranged to provide a focal plane atthe second end of the optical pathway from epifluorescent emission lightreceived from the objective lens. An imaging circuit includes an arrayof optical sensors positioned at the focal plane and configured andarranged to capture an image of the target object from theepifluorescent emission light, the image having sufficient field of viewto capture multiple individual capillary blood vessels and sufficientresolution to distinguish the individual capillary blood vessels fromone another.

Certain aspects of the present disclosure are exemplified in a number ofillustrated implementations and applications, some of which are shown inthe figures and characterized in the claims section that follows. Theabove overview is not intended to describe each illustrated embodimentor every implementation of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

Aspects of the present disclosure may be more completely understood inconsideration of the detailed description of various embodiments of thepresent disclosure that follows in connection with the accompanyingdrawings, in which:

FIG. 1 depicts a block diagram of an epifluorescence microscope device,consistent with an embodiment of the present disclosure;

FIG. 2 depicts a block diagram of an epifluorescence microscope devicewith an external optical source, consistent with an embodiment of thepresent disclosure;

FIG. 3 shows a cross-section of a miniature fluorescence microscope,consistent with an embodiment of the present disclosure;

FIG. 4 depicts an objective lens and ray propagation therein, consistentwith an embodiment of the present disclosure;

FIG. 5 depicts an optical ray trace diagram of an imaging pathway withtwo lens elements and an additional spectral-filtering components,consistent with an embodiment of the present disclosure; and

FIG. 6 depicts a block diagram for a microscope system, consistent withan embodiment of the present disclosure.

While the present disclosure is amenable to various modifications andalternative forms, specifics thereof have been shown by way of examplein the drawings and will be described in further detail. It should beunderstood, however, that the intention is not to limit the disclosureto the particular embodiments described. On the contrary, the intentionis to cover all modifications, equivalents, and alternatives fallingwithin the spirit and scope of the present disclosure.

DETAILED DESCRIPTION

The present disclosure is believed to be applicable to a variety ofdifferent types of devices and processes, and the present disclosure hasbeen found to be particularly suited for epifluorescent imagingapplications. While the present disclosure is not necessarily limited tosuch applications, various aspects of the present disclosure may beappreciated through a discussion of various examples using this context.

Consistent with certain example embodiments of the present disclosure,epifluorescent imaging is facilitated through the use of a microscopedevice and system. For instance, particular aspects of the device and/orsystem allow the use of ultra-low levels for excitation light, which areused to generate epi-fluorescence in a target object or cell. Someaspects allow for imaging of a large field of view with a highresolution. Still further aspects are directed toward the high-speedcapture of images, which can be viewed in real-time or near real-time.While these points of facilitation are not limiting, they are relevantto a number of different embodiments of the present disclosure.

A particular aspect relates to the proximity between an optical sourceof excitation light and the target object or cell for imaging. Forepifluorescent imaging, the interaction between the excitation light andthe target object causes the generation of imaging fluorescence. Theexcitation light is directed toward the target object and has a specificwavelength configured for absorption by fluorophores, fluorescentmarkers or fluorescent probes. The fluorophores then emit light atdifferent (e.g., longer) wavelengths. The amount of absorbed light isrelated to the amount of excitation light delivered to the targetobject. In this manner, the amount of fluorescence generated iscorrelated to the amount of excitation light. Although various lightdelivery mechanisms can help reduce the attenuation of light as ittravels through a medium, the attenuation of light will increase asdistance of travel through a medium increases. Also, when using air andother mediums, the composition of the medium and other dispersiveattributes can play significant roles in the delivery and/or attenuationof the light, whereas the reduction of the optical path length (mainlyresulting in the reduction of travel of light through air) does next tonothing to decrease attenuation. The design of the microscope device andsystem allows for the placement of the optical source of the excitationlight in close proximity to the target object, thereby facilitating theuse of a short optical path. This is particularly useful forfacilitating the use of an optical source of low power and/or capturingimages using low-levels of light.

Various fluorescence sources can be used consistent with one or moreembodiments discussed herein. The mention of a particular source offluorescence does not necessarily preclude use of other sources offluorescence (e.g., genetically-encoded fluorescent proteins, such asGFP, GCaMP, and variants thereof).

Other aspects of the present disclosure relate to the integration ofoptics, filters, and camera into a single housing, which can beparticularly useful for the elimination of the fiber-bundle and all ofits associated limitations.

Yet other aspects relate to the proximity of a target object or cellrelative to an image sensor for capturing image data from epifluorescentlight. Image resolution and imaging times are related to the amount ofepifluorescent light that can be collected and detected by an imagesensor. Attenuation of the epifluorescent light due to properties of theoptical path between the target object and the image sensor can beundesirable. Careful design of the microscope device and system allowsfor placement of the image sensor in close proximity to the targetobject, thereby facilitating the use of a short optical path.

Also in accordance with the present disclosure, the proximity of anobjective lens of a microscope device is set relative to a targetobject, during imaging of the target object. Large distances between anobjective lens and the target object can have a detrimental effect onthe amount of the excitation light received at the target object as wellas the amount of fluorescence received at and collected by the objectivelens. Accordingly, setting the proximity of the object lens relative tothe target object can be advantageous.

Embodiments of the present disclosure relate to a microscope device andsystem that captures image data for a relatively large field of view,the image data providing high resolution of a target object. One suchembodiment of the present disclosure includes an image capture circuit,with an array of sensor elements or pixels, which is provided to imagethe field of view. The sensor elements detect epi-fluorescence fordifferent portions of the field of view. The sensor elements can beconfigured with sufficient sensitivity and proximity to the targetobject to facilitate image capture and generation.

Other embodiments of the present disclosure relate to the length ofexposure times for image capture. As fluorophores are excited, they canbegin to lose their ability to fluoresce, which is sometimes referred toas photobleaching. Moreover, epi-fluorescence imaging involves theabsorption of the excitation light by the target object. Some of thisabsorbed light is converted into heat. This generated heat can placelimits on the exposure time, e.g., the heating of biologicalmaterial/cells can cause cell damage and even death. The exposure time,however, can be increased if the intensity of excitation light isdecreased. The intensity of the excitation light can be reduced if, forexample, the optical coupling between the target object and the imagesensor is improved. Phototoxic effects can be more damaging thanlocalized heating. Aspects of the present disclosure lessen or eliminatethese effects which adversely impact image capture and relatedprocessing of the data.

Particular embodiments of the present disclosure relate to theadjustment of excitation light intensity in conjunction with theadjustment of exposure time to improve image quality, or an image for aparticular goal (e.g., image capture rate, resolution, field of viewsize or imaging depth).

According to other aspects of the present disclosure, relatively lowoptical zooms are used in connection with high-resolution imaging of afield of view for target objects of small size. Constraints on theoptical zoom required for a particular level of imaging can be lessenedthrough the careful design and application of a microscope device andsystem consistent with various aspects discussed herein.

Embodiments of the present disclosure relate to the real-time imaging oftarget objects using a microscope device and/or system consistent withaspects discussed herein. In certain of these embodiments, the imagingrate is increased by reducing the field of view while holding a constantresolution, the image capture time is reduced by reducing the exposuretime and/or the frame rate achievable for such real-time imaging iscorrelated to size of the full field of view as well as the desiredimage resolution. Another factor optionally implemented therewithincludes the type and responsiveness of the image sensor that is used.Still other factors relate to the ability to transmit and process theimage data for display, should it be desirable to view the images inreal-time.

Still other embodiments of the present disclosure relate to thefacilitation of in vivo or in vitro epifluorescent imaging. Forinstance, in vivo imaging of a live subject can be particularly usefulfor correlating external stimuli and other factors with the capturedimages. This correlation can be used, for example, as adiagnostic/research tool by associating properties of the capturedimages with the external stimuli. Real-time imaging at high frame ratescan further provide such correlation as a function of time.

An embodiment of the present disclosure is directed toward a microscopedevice and/or system having a modular design that facilitates detachingand reattaching various components of the microscope device. Thedetachment and reattachment can be used to replace the modularcomponents with new and/or different modular components. For instance,the light source can be replaced with a new light source having the sameor different optical and electrical properties. The array of opticalsensors and/or the optical direction elements (e.g., mirrors, filtersand lenses) can also be removed and replaced. If desired, the opticalsensor can also be removed and replaced.

In certain other embodiments consistent with the instant disclosure, oneor more of the imaging devices includes a synchronization circuit forinterfacing to an external optical-data processing (recording and/orconfiguring) system. The synchronization circuit includes logiccircuitry (e.g., a programmable or semi-programmable chip(microcontroller or ASIC) that is configured and arranged to communicatea frame reference/active signal. In a typical application, a frameactive signal would provide synchronization information, e.g., asdefined in an IEEE communications standard, for and with the datacommunicated between the imaging device and the external system. Such anoptical-data recording/configuring system can be used to installsoftware, configure set-up parameters for experiments and procedures,provide visual feedback during such experiments and procedures, andrecord the optical data for manipulation and further study.

In yet further embodiments, the instant disclosure is directed tomethods of using the image devices which are described herein. Certainof the devices include a base plate acting as a foundational structurewhich provides support/stability and also allows for microscope(re)alignment. These methods include the steps of attaching andreattaching the epifluorescence microscope to the base plate forallowing the microscope alignment to be precise. Such precision shouldbe sufficient for repeated imaging of a common imaging location, e.g.,during chronic experiments.

Turning now to the figures, FIG. 1 depicts a block diagram of anepifluorescence microscope device, consistent with an embodiment of thepresent disclosure. The epifluorescence microscope device 100 includes anumber of components within the dimensions 120 and 122. Not shown is afurther dimension, which extends perpendicular to the dimensions 120 and122. Although not necessarily limited thereto, each of these dimensionscan be less than an inch. Consistent with other embodiments, thedimensions are slightly larger, e.g., on the order of a few centimeters.

The epifluorescence microscope device 100 includes an optical source102. This optical source 102 generates excitation light 104. In aparticular implementation, the optical source 102 is alight-emitting-diode (LED) or an organic light-emitting-diode (OLED).The excitation light 104 is directed by an optical arrangement 124 to atarget object 114, for imaging thereof. The optical arrangement caninclude one or more of objective lens 112, (dichroic) mirror 110 andexcitation filter 108 and an emission filter (not depicted).Epifluorescent light 116 from the target object 114 is directed from/bythe objective lens to an image capture circuit 118. The epifluorescencemicroscope device 100 is configured to direct light from and captureimage data for a field of view 126.

In various embodiments of the present disclosure, the microscope device100 can also include one or more of an image-focusing optical element(e.g., an achromatic lens) and an emission filter. These and otherelements can help control optical properties of the microscope device100.

Consistent with one embodiment, the depicted elements are eachintegrated into a relatively small area, e.g., within a single housinghaving dimensions 120, 122. Such integration of the various componentscan be particularly useful for reducing the length of the opticalpathway from the optical source 102 to the target object 114 and back tothe image capture circuit 118. The reduction of this optical pathway canbe part of the configuration parameters that facilitate a number ofdifferent properties and capabilities of the microscope device 100. Forexample, in certain embodiments the microscope can provide images with aresolution to 1 um for an imaging field of view of up to 1 mm² in area.

A particular example embodiment is configured with an array of opticalsensors 118. An optical arrangement 124 is configured to directexcitation light 104 of less than about 1 mW (various embodimentsprovide for a higher excitation power, e.g., 100 mW) to a target object114 in a field of view 126 of that is at least 0.5 mm² and to directepi-fluorescence emission 116 caused by the excitation light 104 to thearray of optical sensors 118. In various embodiments, the field of view126 can be at least 1 mm². The optical arrangement 124 and array ofoptical sensors 118 each configured sufficiently close to the targetobject 114 to provide at least 2.5 μm resolution for an image of thefield of view 126. In other embodiments, the optical arrangement 124 andarray of optical sensors 118 can be configured to provide at least 1 μmresolution. In certain embodiments, the excitation optical power at thespecimen is variable and can be in the range of 100 μW-100 mW, dependingupon the particular configuration and imaging constraints.

Consistent with an embodiment of the present disclosure, the opticalsource 102 can deliver light of up to 37 lumens or 6 mW. It is not,however, necessarily a requirement that the optical source 102 providelight of such intensity. Moreover, the amount of light received by thetarget object is less than (relative to an attenuation factor) theamount of light provided by the optical source 102. For instance, theattenuation of one embodiment results in 6 mW at the light sourcecorresponding to 1 mW excitation power delivered at the target object.Similarly, to deliver 100 mW of excitation power at the specimen thelight source can be configured to provide up to 600 mW.

Although FIG. 1 depicts the various components as being within thedimensions 120, 122, other embodiments are possible. For instance, FIG.2 depicts a block diagram of an epifluorescence microscope device withan external optical source, consistent with an embodiment of the presentdisclosure. The epifluorescence microscope device 200 includes anexternal optical source 214. This external optical source 214 is coupledto an optical arrangement 250 that includes a number of componentswithin the dimensions 216 and 218. Not shown is a further dimension,which extends perpendicular to the dimensions 216 and 218. Although notnecessarily limited thereto, each of these dimensions can be less than acubic inch. Consistent with other embodiments, the dimensions are on theorder of a few centimeters.

Consistent with one embodiment of the present disclosure, the externaloptical source 214 is coupled to the optical arrangement 250 via a fiberoptic cable 212. Excitation light from the external optical source 214and the fiber optic cable 212 pass through (optional) excitation filter208. A (dichroic) mirror 204 and objective lens 206 direct theexcitation light to the target object 210. In particular, the excitationlight is directed toward field of view 220. The excitation light causesfluorophores in the target object 210 to fluoresce with epifluorescentlight. This epifluorescent light is directed by (dichroic) mirror 204and objective lens 206 to optical sensor 202.

In various embodiments of the present disclosure, the microscope device200 can also include one or more of an image-focusing optical element(e.g., an achromatic lens) and an emission filter in the imagingpathway. These and other elements (not shown FIG. 1 ) can help controloptical properties of the microscope device 200.

Although the optical source 214 is not located in close proximity to theoptical arrangement 250, the amount of excitation light that isdelivered to the target object 210 can still be set at a low level dueto the proximity between the target object 210, the objective lens 206and/or the optical sensor 202. In particular, this close proximity canbe particularly useful for providing efficient optical coupling betweenthe target object and the optical sensor. Thus, the epi-fluorescence canbe of a lower intensity relative to the image properties. Moreover, alower level of excitation intensity at the target object 210 can allowfor longer exposure to the excitation light before photobleaching,heating or other adverse effects become a factor.

The following discussion provides details of an experimental embodiment.Although the experimental embodiment provides examples and detailsregarding various parameters and results, these aspects are notnecessarily limiting to the various other embodiments of the presentdisclosure. The experimental embodiment was configured and arranged toprovide a small epi-fluorescence microscope. The microscope included aspecially-integrated arrangement that included the light source, optics,filters, and camera into a single housing.

The level of integration and the resulting size scale for the miniaturefluorescence microscopes can be configured for use in a multitude ofapplications. A particularly challenging application relates to in vivobrain imaging, e.g., in a mouse or similar organism. In at least onesuch application, the microscope is designed to be mounted on the headof a mouse for in vivo brain imaging during awake behavior. In order tobe configured for this and other applications, the microscope wasdesigned with stringent physical size and mass requirements, e.g., so asto be easily borne by the mouse during awake and active behavior. Forinstance, given that an adult mouse is approximately 25 g in mass, themicroscope was designed to be 3 g or less. Other design considerationsrevolved around the image quality, reliability and speed.

One embodiment was configured for imaging of high-speed, cellular-levelbrain imaging. The cost and simplicity of large-scale manufacturing wasanother factor in the design of the fluorescent microscope. Particularembodiments were configured and designed as an integrated device thatwas mass-producible at low costs (e.g., scalable and amenable tomass-production).

FIG. 3 shows a cross-section of a miniature fluorescence microscope thatwas designed consistent with such considerations and other embodimentsof the present disclosure. The vertical arrows denote the excitation(down arrow) and emission (up arrow) pathways. A single housing 300contains the light source 314 and image capture circuit 302, as well asa fluorescence filter set (emission filter 306 and excitation filter316) and microoptics (collector lens 312, dichroic mirror 310,achromatic lens 308, objective lens 318 and focusing mechanism 304).This integration of the light source and camera with the filter set andmicroscope optics facilitates high-resolution image capture in variousapplications, such as in vivo imaging.

Consistent with one embodiment, a solid-state light-emitting-diode(LED), which is small, amenable to integration with collection optics,and mass-producible at low costs, is used for the excitation lightsource. A Complementary-Metal-Oxide-Semiconductor (CMOS) image sensor isused for the camera.

In a particular experimental embodiment of the present disclosure, theLED light source shown in FIG. 3 can be implemented using a blue LED 314mounted on a custom 6 mm×6 mm printed circuit board (PCB), which alsoincludes a heatsink. A drum micro-lens 312 is used to collectillumination, which then passes through a 4 mm×4 mm excitation filter316, deflects off a dichroic mirror 310, and enters the imaging pathway.A gradient refractive index (GRIN) objective micro-lens 318 focusesillumination onto the sample. Fluorescence emissions from the samplereturn through the objective 318, the dichroic 310, a 4 mm×4 mm emissionfilter 306, and an achromatic doublet tube lens 308 that focuses theimage onto the CMOS image sensor 302 (640×480 pixels), mounted on a 8.4mm×8.4 mm PCB with power and signal conditioning electronics. The LEDlight source, CMOS camera, and the optical components are integratedinto a microscope housing 300 with a modular design that permitsindividual components, such as the excitation LED and CMOS camera chip,to be replaced for different application needs. Moreover, a memorycircuit can be integrated to store image data. The modular aspect allowsthe memory circuit to be removed and replaced without removing themicroscope from the imaging target (e.g., the microscope could remainaffixed to an organism). Thus, captured images are stored locally andthen retrieved by removal of the memory circuit, which can be configuredto interface with an external device, such as a laptop computer.

In example embodiments, the microscope housing is fabricated usingPolyetheretherketone (PEEK) and has built-in mechanical image focusingcapabilities permitting focusing to sub-micron accuracy by adjustment ofthe camera position. Other materials (e.g., bio-compatible andsolvent-resistant materials) can also be used consistent with variousdesired applications. The microscope can be plugged into a computer viaexternal data acquisition PCBs, with a standard USB interface, providingreal-time image acquisition, display, and camera and light sourcecontrol.

Embodiments of the present disclosure are directed toward the design andcontrol over an imaging pathway and design of an epifluorescencemicroscope. The imaging pathway includes an objective lens along withother optical conditioning and directing components. The additionalcomponents can include, for example, spectral-filtering componentsand/or an achromatic doublet imaging tube lens.

FIG. 4 depicts an objective lens and ray propagation therein, consistentwith an embodiment of the present disclosure. In a particularembodiment, the objective lens 402 is a GRIN objective lens. The GRINobjective lens is a cylindrical lens with a radially-decreasingrefractive index profile that results in rays 406, originating fromtarget object 404, propagating in a sinusoidal path, as shown in FIG. 4. A GRIN lens can be particularly useful due to the small form factorand ease-of-integration with other microoptics and/or for reducingoptical path length relative to other types of objective lenses.

In one experimental embodiment of the present disclosure, the GRINobjective lens used to collect fluorescence emission from the specimenis 2 mm in diameter with a pitch length of 0.245. A pitch length of 1corresponds to one full sinusoidal path of ray propagation; thus a pitchlength of 0.245 results in light rays that are close to being consideredcollimated light rays, as shown in FIG. 4 . The objective numericalaperture (NA) is 0.45. Collected fluorescence emission is passed throughthe dichroic mirror and the miniature emission filter, and thefluorescence image is then focused by an achromatic lens, with a focallength of 15 mm, onto the CMOS image sensor.

FIG. 5 depicts an optical ray trace diagram of an imaging pathway withthe two lens elements and the additional spectral-filtering components,consistent with an embodiment of the present disclosure. The rays showhow points in the specimen plane are imaged onto the CMOS camera. Lightrays (502, 504, 506, 508, 510) are traced from five distinct pointsources in the specimen plane to imaged points on the CMOS camera. Thedesign of the imaging pathway and the optical ray trace simulations wereperformed using software modeling. The light rays emanating from targetobject 512 pass through the GRIN objective lens 514. The GRIN objectivelens 514 collimates the light rays. The light rays are then directed bydichroic mirror 516 toward achromatic lens 518. Emission filter 520filters out undesired light wavelengths, such as reflected excitationlight. The light rays then strike sensor array/camera 522, where theyare recorded and used to generate an image of the target object 512.

The optical magnification provided by the imaging pathway and opticalelements can be configured accordingly to the desired application.Moreover, the need for optical magnification can be offset by theproximity of the objective lens to the target object as well as theproximity between the target object, objective lens and the imagecapture circuit, resulting in embodiments where low opticalmagnifications (1-4×) can permit imaging large specimen fields-of-viewgreater than 1 mm² while still providing high spatial resolution of atleast 1 μm.

Consistent with experiments and related embodiments, the microscopeoptical magnification range is between 4.5-5.5×. The working distance,that is, the distance from the near surface of the objective to thepoint in the specimen plane that is in focus, is about 150-200 μm orabout 50-250 μm (these dimensions can depend on the exact positioning ofthe focal plane). The performance of an optical design can be evaluatedby its resolving capabilities, and one measure of this is thefull-width-half-maximum (FWHM) of the optical point-spread function. Theon-axis, lateral spatial resolution of the imaging pathway computed inthis manner was approximately 1.2 μm, degrading to approximately 1.6 μmat the periphery of the field-of-view. This measurement, however, is notnecessarily limiting as the spatial resolution achievable is also afunction of various factors including, but not limited to, the camerapixel size.

Aspects of the present disclosure relate to properties of theillumination pathway between the target object, the excitation sourceand the image sensors. For instance, careful design of the illuminationpathway can provide efficient and uniform excitation of the specimenunder observation. The coupling of the excitation light source to theillumination pathway can be useful for providing sufficient andwell-controlled illumination to excite the specimen. In one experimentalimplementation, a blue LED with the spectral peak of illumination ataround 470 nm was used as the excitation light source. The LED wasmounted on a 6 mm×6 mm PCB that was equipped with a heat sink. The heatsink helps to keep the LED junction temperature stable during operation.

LED illumination output is (first order) linear as compared with drivecurrent only over a local area (the actual transfer function is acurve). However, the output exhibits temperature dependence. Theexperimental results showed that drive currents of 20-30 mA weresufficient to deliver the required illumination power at the specimen.This drive current was approximately one fiftieth ( 1/50) of the maximumrating for the drive current of the LED (e.g., maximum drive current is1 A and typical drive currents are 20 mA). For a given drive current,the LED junction generally reached an equilibrium temperature inapproximately 60 s after LED turn-on, and the LED illumination outputstabilized. In certain embodiments, the LED light output can bestabilized in real-time over temperature variations via intrinsic orexternal temperature measurement coupled with a feed-forward or feedbacksystem. For instance, data received from a temperature sensor (e.g.,temperature sensitive resistor or temperature sensing diode) and/orcurrent sensor can be used to control the amount of electrical powerprovided to the LED. In certain embodiments, a control circuit forproviding such control can be calibrated during manufacturing or at apoint thereafter.

Consistent with an experimental embodiment, the LED illumination iscollected by a drum lens, passed through a miniature fluorescenceexcitation filter, and then reflected off a dichroic mirror that directsthe illumination into the GRIN objective lens and to the specimen. Thesystem was designed for collection and delivery of light to the specimento achieve spatially uniform, homogenous illumination at an averageoptical power density across the specimen field-of-view. This can beaccomplished by approximating Kohler illumination. In Kohlerillumination, the light source and the specimen planes are on separatesets of conjugate planes, ensuring that the light source is not imagedonto the specimen, and yielding even illumination of the specimen at anaverage optical power density.

According to an experimental embodiment, the fluorescence filter set isconfigured to separate the excitation illumination from the fluorescenceemission. The filter set includes three parts: the excitation filter,dichroic mirror, and emission filter. The spectral profiles of thefilters and dichroic were configured to allow blue excitation and greenemission. These spectral profiles are amenable to imaging a broadpalette of synthetic fluorescent probes, such as fluorescein and itsreactive derivatives, as well as genetically-encoded fluorescentproteins, such as the green fluorescent protein (GFP). For a particularexperimental implementation, the specific spectral characteristics anddimensions of the filter set were as follows. The excitation filter wasa bandpass filter with a spectrum of 480/40 nm and a dimension of 4 mm×4mm×1.05 mm, the emission filter was also a bandpass filter with aspectrum of 535/50 nm and a similar dimension of 4 mm×4 mm×1.05 mm, andthe dichroic mirror had a long pass spectral profile, passingwavelengths above 506 nm, and with a dimension of 4 mm×4.8 mm×1.05 mm.In other embodiments, the filter set can be configured to permitmultiple wavelength excitation for excitation and imaging of multiplefluorescent markers with different excitation/emission spectra.

Embodiments of the present disclosure are directed toward the use of aCMOS image sensor. CMOS image sensors are digital imaging sensors thatare designed and fabricated in CMOS. This can be particularly useful forproviding image sensors that can be mass-produced at low costs.Moreover, the use of CMOS technology can be useful for providing asolution that operates at both low power and at high speed. The CMOSimage sensors can be implemented with digital pixels, where conversionfrom photons to bits is done directly at the pixel level with aper-pixel analog-to-digital converter and dynamic memory. This can beparticularly useful for high speed imaging applications and theimplementation of still and video rate imaging applications that benefitfrom high-speed capture, such as dynamic range enhancement.

In a particular implementation a CMOS image sensor was used that had aresolution of 640×480 pixels, each pixel having dimensions of 5.6 μm×5.6μm. The CMOS image sensor was packaged in a 5.6 mm×5.8 mm chip-scalepackage. The sensor output was in a serialized digital low-voltagedifferential signaling (LVDS) format. Such a LVDS format is particularlyuseful for facilitating the interfacing with a minimum number ofinterconnects, which can be an important consideration for minimizingthe number of wires attached to the microscope.

Experimental characterizations, shown in table 1, of the sensor arebriefly described as follows. Pixel read noise was estimated bycalculating the standard deviation of pixel intensity in 1000 imageframes, acquired in full darkness and with sufficiently brief exposuresuch that the noise contribution from dark current shot noise wasinsignificant. Dark current, and dark signal non-uniformity (DSNU), thevariation in dark current across the array of pixels due to devicemismatches, were estimated by capturing 1000 frames in the dark withsufficiently long exposure times, and then averaging the frames into asingle image, with the objective of ideally averaging out temporalnoise. Dark current and dark signal non-uniformity were then found fromthe mean and standard deviation of the pixels in the averaged image.With these experimentally-characterized sensor specifications, and otherknown electronic properties of the sensor, the CMOS image sensor wasanalytically modeled to estimate imaging fidelity for a range ofincident photon flux densities.

TABLE 1 Package size 5.6 × 5.8 mm² Array size 640 × 480 pixels Pixelsize 5.6 × 5.6 μm² Frame rate 36 fps/Hz Pixel read noise 10 e⁻ Darkcurrent (room temp.) 900 e⁻/s Dark signal non-uniformity 30 e⁻/s Fullwell capacity 52,000 e⁻

The experimental results are illustrative and not meant to be limiting.For instance, the frame rate/image capture speed of Table 1 (36 Hz) isto be understood in the context of the specific experimental parameters.For instance, the captured field of view (FOV) was at least 0.5 mm²,although it could be up to 1 mm² or even more. Smaller FOVs would allowfor higher frame rates (e.g., 370 μm 370 μm at 100 Hz).

One application consistent with embodiments of the present disclosurerelates to in vivo mouse brain imaging experiments. Since photon fluxdensities incident on the sensor plane for typical in vivo mouse brainimaging experiments are on the order of 10¹¹ photons/cm²/sec, whichcorresponds to 20,000 electrons/pixel/sec, the CMOS image sensoroperates in the photon shot noise limited regime for in vivo mouse brainimaging experiments. Thus, the CMOS image sensor's pixel read noise anddark current numbers, relevant considerations for applications whereimaging is performed in low-light conditions, have a negligible impacton imaging fidelity. Along with an estimated sensor dynamic range of 60dB, which is believed to be more than sufficient for capturing the rangeof signal intensities observed in in vivo brain imaging datasets, theimaging performance metrics of the CMOS image sensor were shown to bewell-suited to serving the application needs.

Embodiments of the present disclosure relate to communication of imagedata, control signals and/or power to the microscope device. For manyapplications, the intrusiveness of the microscope is a relevantconsideration. This aspect can be adversely affected by the number ofwires used to provide the communication and/or power to the microscopedevice. Accordingly, various aspects of the present disclosure aredirected toward reducing the number of wires between the microscope andan external system, which can provide control and/or image storage andprocessing functions. Consistent with a particular experimentalimplementation, a two-wire I2C interface is used to communicate controlinformation with the microscope device. The I2C interface defines thewires as SCLK and SDATA and communicates using a serial interface,thereby providing a low wire count solution. In certain embodiments, anadditional rotational element (e.g., commutator) can be used tofacilitate movement and to lessen or eliminate torsional strain on theconnection wires. Various other protocols and communication solutionsare possible.

Consistent with a particular embodiment of the present disclosure, theinput power supply is stepped-down and regulated by a low-dropoutvoltage regulator (LDO) before being delivered to the image sensor. Aninput clock signal (162 MHz) is transmitted to and restored by a clockbuffer before being sent to the image sensor. The received clock signalis then used to internally generate a 27 MHz master clock signal. Theimage data output of the sensor is in a 10-bit digitized format andtransmitted over a two-wire serial LVDS protocol. The presentdisclosure, however, is not necessarily limited to any particularcommunication protocol or power providing mechanism.

FIG. 6 depicts a block diagram for a microscope system, consistent withan embodiment of the present disclosure. Two of the electronicallyactive components of the microscope 600 include the optical excitationsource 602 and the sensor array 604. In certain embodiments, themicroscope 600 receives power and control signals from an externalinterface module 650. This allows various circuits and components (e.g.,power supply, memory storage and/or image processing) to be locatedremote from the microscope. Interface module 650 can be designed tofunction as a stand-alone component or for connection with anotherdevice, such as a computer.

In certain embodiments, interface module 650 is configured to providemicroscope data acquisition and control and is external to themicroscope imaging device. In another embodiments, interface module 650(with or without input/output (I/O) interface 616) can be integratedwith the microscope device 600, e.g., for applications where theweight/size does not preclude such integration.

According to one embodiment of the present disclosure, the interfacemodule 650 includes an input/output (I/O) interface 606 (atransmitter/receiver/transceiver circuit). This I/O interface 606 can beused to provide power, control and to transmit image data to and fromthe microscope 600. For instance, power can be provided from one or morepower regulators 610; control signals can be provided from a controlinterface 614; driver signals 608 for powering the optical excitationsource 602; and image data can be communicated to an (image) dataprocessing unit or circuit 612. Accordingly, microscope 600 can also beconfigured with one or more transmitter/receiver/transceiver circuits toallow for communication with the interface module 650.

In one embodiment of the present disclosure, I/O interface 606 isconnected to the microscope 600 using wired connections. The wiredconnections can transmit power and communication signals using anynumber of different protocols. Particular applications (e.g., in vivoimaging of active organisms) benefit from the wired connection beinglight, flexible and otherwise amenable to movement by the object of theimaging. Thus, certain embodiments implement communication protocols andsolutions with low pin/wire counts.

Consistent with other embodiments of the present disclosure, I/Ointerface 606 is designed to use wireless communications. Wirelesscontrol of the microscopy imaging device and wireless data transfer canbe particularly useful when several moving imaging objects are beingimaged in parallel in sufficiently close proximity to each other. Inone, non-limiting, instance, I/O interface 606 can use magnetic fieldinduction, such as near-field communications derived from ISO/IEC 14443.Near-field communications also allow for power to be provided to themicroscope wirelessly, e.g., through inductive coupling. Other wirelesscommunication protocols and solutions are also possible.

Consistent with various embodiments, the interface module 650 isdesigned with an input/output (I/O) interface 616 that interfaces withanother device, such as a laptop/desktop computer. This I/O interface616 could also include a display screen for presenting images capturedfrom microscope 600. Consistent with certain embodiments, I/O interface616 can be integrated as part of the interface module 650 or a separatecomponent (e.g., connected via wired or wireless communication links).

The various examples discussed herein for the I/O interfaces 606 and 616are not limiting. The I/O interfaces can be custom designed orimplemented consistent with existing communication protocols.

In certain embodiments, memory 618 can be used to store image dataand/or software instructions for execution by data processing unit orcircuit 612, which can be implemented using specialized processor (e.g.,field programmable gate arrays) or general purpose microprocessorsconfigured to execute the specialized software instructions. The memory618 can include circuits providing non-volatile memory (e.g., flash)and/or volatile memory (e.g., volatile random access memory (RAM)).

A specific embodiment of the present disclosure is implemented using twoprinted circuit boards (PCBs) that are contained within the microscope600. The first PCB 602 includes a light emitting diode (LED). The secondPCB 604 includes a complementary metal-oxide semiconductor (CMOS)imaging/camera chip. These PCBs are both connected to a custom externalsystem 650 via nine thin and flexible wires (2 wires to the LED PCB 602and 7 to the camera PCB 604) that are encased in a single polyvinylchloride (PVC) sheath of outer diameter 1.5 mm. The external system 650interfaces with a computer via a general-purpose USB imaging dataacquisition adapter. This configuration can be particularly useful forenabling real-time microscope control and data acquisition as well asimmediate display of images.

An Inter-Integrated Circuit (I2C) serial communication interface isprovided using an I2C controller 614. The I2C interface can be used tocontrol the operation and function of the (CMOS) imaging/camera chipthat is part of PCB 604. The image data output from the imaging/camerachip is serialized and transmitted according to a digital low-voltagedifferential swing (LVDS) format.

Consistent with the various embodiments discussed herein, experimentalfluorescence microscopes can be fabricated, assembled, and tested. Themicroscope fabrication, assembly, and testing processes can beimplemented in a distributed and streamlined manner. Camera and LED PCBscan be fabricated separately, while lenses and filters are produced orprocured independently. The microscope housing can be fabricated as akit of individual parts to facilitate manufacturing thereof.

With or without imaging optics, the camera PCB can be tested for power,camera control, and the presence of valid output data. Testing of theLED PCB can include the driving of the LED while illumination output ismonitored. Once fully assembled, the microscope housing is designed tomaintain the optical parts in alignment with the LED and camera PCBs.The microscope housing was made of black Polyetheretherketone (PEEK),which is lightweight, chemically resistant, stiff, and machinable.Although the black housing absorbed the majority of stray light, a thinlayer of black felt or other absorbent material can be affixed (e.g.,glued) in locations prone to light reflection. A threaded interfacebetween the part of the housing holding the camera PCB and themicroscope body is configured to provide fine adjustment of the spacingbetween the two for setting the specimen plane that is in focus in theacquired image. The modular nature of the microscope design permitsremoving and interchanging various parts as required, for example,camera and LED PCBs and the filter and dichroic set.

Experimental microscopes manufactured consistent with this method weretested for a variety of features. Table 2 depicts various specificationsfor the experimentally-fabricated miniature fluorescence microscope usedfor in vivo imaging of a brain for an active mouse and without imagealignment.

TABLE 2 Dimensions 8.4 × 13 × 22 mm³ Mass 2 g Resolution 2.5 μmField-of-view 0.48 mm² Photon flux 3 × 10¹¹ ph/cm²/s SNR 37 dB Imagingduration 40-50 mins

Simulated microscope resolution, based on the modulation transferfunction (MTF) of the microscope, was determined to be 2.3 μm. Measuredmicroscope resolution, as stated in Table 2 above, was empiricallyestimated to be approximately 2.5 μm. Microscope resolution was measuredby imaging a Siemens Star resolution test pattern.

In order to test the resolution capabilities of the experimentalmicroscope, a sharp edge, a slanted bar, was used as the synthetic sceneand imaged with the virtual microscope. The average edge response, orline spread function, was then derived at different cross-sections ofthe digital image of the slanted bar and the MTF was then calculated.The results support that the Nyquist rate, as determined by the camerapixel pitch, was found to be 89 cycles/mm. This corresponds to a 2.2 μmfeature size in the specimen plane. The MTF10, that is, the resolutionat which the contrast degrades to 10% of the ideal contrast was shown tobe 2.3 μm.

A number of variations are possible from the expressly-discussedembodiments of the present disclosure. For instance, the microscope canbe configured to include a local power supply, such as a battery. Inother instances, an array of microscopes can be arranged to capturerespective images of target objects.

Particular embodiments relate to in vivo imaging of an organism. Variousembodiments discussed hereafter relate to the imaging of cerebellarvermis to study microcirculation concurrently with locomotive, and othermouse behaviors, by mounting of an integrated microscope on the cranium.Notwithstanding, the present disclosure is not so limited and can beapplied to a variety of different fields and applications.

In particular experimental embodiments relating to in vivo imaging,brain imaging, with the miniature microscope fixed over the mouse brain(in multiple experiments), was implemented once for a mouse exhibitingvigorous locomotor activity. The microscope was attached while the mousewas anesthetized and imaging commenced about 15-60 min after removalfrom the anesthesia. Using the cranially-attached microscope, multiplevideo clips of mouse behavior and the correlated microcirculation in thevermis can be captured for various behaviors. For instance, the mousewalking about the behavioral arena represents a first behavior, whilethe mouse running on an exercise wheel represents a second behavior.

In an experimental implementation, microcirculation was recorded usingthe integrated microscope that captured images at 100 Hz following anintravenous injection of FITC-dextran. This fluorescent dye labeled theblood plasma, allowing erythrocytes to be seen in dark relief.Individual erythrocytes were witnessed flowing through the capillaries.To reduce the possibility of photo-induced alterations in physiology,the duration and mean power of continuous illumination was limited to <5min and <600 μW for each imaging session. At least 2 min were allowed toelapse between imaging sessions, and the total imaging duration over thecourse of an experiment was generally around 45 min. Frame acquisitionrates were around 100 Hz for the cerebellar vasculature andmicrocirculation imaging experiments, and 30-46 Hz for Calcium imagingstudies.

Although several in vivo applications are discussed herein, the devicesand methods of the present disclosure can be used for other imagingsolutions, such as morphology determinations, drug screening and otherapplications.

Consistent with one embodiment, the use of integrated microscopesdiscussed herein facilitates the identification of phenotypes forvarious organisms. This is facilitated by the high-resolution imagingthat can be used to identify distinguishing characteristics. Forinstance, phenotypes can be identified for wild-type and erbb3 mutantzebrafish with fluorescence immunolabeling of myelin basic protein withAlexa-488. The spinal cords and posterior lateral nerve can be imagedand used to distinguish in wild-type fish. In erbb3 fish Schwann cellsfail to develop the posterior lateral nerve.

Consistent with another embodiment, the use of integrated microscopesfacilitates accurate cell counting assays in well plates. For instance,a base concentration (CO≈4.0×105 cells/mL) of live MCF7 human breastcancer cells labeled with carboxyfluorescein can be diluted, with 8sample wells for each of 6 concentrations. Optionally, an automatedalgorithm can be used to provide for fast and efficient counting ofcells in the images.

Consistent with one embodiment, the automated algorithm uses successivestages of analysis within a custom cell counting algorithm. A logiccircuit such as a (computer) processor circuit (e.g., including a memorycircuit/medium for providing the process instructions) performs contrastequalization on (raw) fluorescence image of live MCF7 human breastcancer cells labeled with carboxyfluorescein. The processor circuit nextconverts the resulting image to binary format, to which an initialsegmentation is performed. Single cells are then identified and counted.Iterative rounds of morphological filtering allow segmentation of theclusters of multiple cells that remained after the initial segmentationinto individual cells.

Embodiments of the present disclosure are directed toward using amicroscopy imaging device as part of a larger optical system. Forinstance, a microscopy imaging device can be embedded in vivo tofacilitate long-term, chronic imaging. This can be facilitated byproviding mobile power sources and control/processing circuitry. Theseand other elements can be integrated within the housing of themicroscopy imaging device or connected externally (e.g., using a wiredconnection to a control unit located elsewhere on the subject). Inanother instance, a microscopy imaging device can be used in connectionwith specialized optical devices, for example, to facilitate in vivoendoscopy or to monitor the subject during surgical procedures.

The various embodiments described above and shown in the figures areprovided by way of illustration only and should not be construed tolimit the disclosure. Based on the above discussion and illustrations,those skilled in the art will readily recognize that variousmodifications and changes may be made to the present disclosure withoutstrictly following the exemplary embodiments and applicationsillustrated and described herein. For instance, applications other thanin vivo imaging may be amenable to implementation using similarapproaches. In addition, one or more of the above example embodimentsand implementations may be implemented with a variety of approaches,including digital and/or analog circuitry and/or software-basedapproaches. These approaches are implemented in connection with variousexample embodiments of the present disclosure. Such modifications andchanges do not depart from the true scope of the present disclosure,including that set forth in the following claims.

As discussed above, specific applications and background detailsrelative to the present disclosure are discussed above, in thedescription below and throughout the references cited herein. Theembodiments in the Appendices may be implemented in connection with oneor more of the above-described embodiments and implementations, as wellas with those shown in the figures and described below. Reference may bemade to the Appendices filed in the underlying provisional application,which are fully incorporated herein by reference.

1-4. (canceled)
 5. A method comprising: upon removably connecting anepifluorescence microscopy system, which has a dimension not exceeding 1inch along an axis parallel to a specimen plane, to a support structurethat has been affixed to a target object, directing first light from alight source through an optical excitation arrangement of theepifluorescence microscopy system towards a pre-defined area at thespecimen plane in the target object; with an imaging circuit of themicroscopy system that includes an optical sensor array, receivingfluorescence generated at the pre-defined area as a result of saiddirecting to generate image data; and with a synchronization circuit ofthe microscopy system, comparing said image data with informationrepresenting visual feedback generated outside the epifluorescencemicroscopy system to transform parameters of said directing first light,wherein the synchronization circuit is in operable communication withthe imaging circuit.
 6. A method according to claim 5, wherein saiddelivering the first light includes delivering the first light having anoptical characteristic sufficient to optically identify Ca2+-spikingconcurrently in up to >200 Purkinje neurons.
 7. A method according toclaim 5, wherein said receiving fluorescence includes receivingfluorescence through a combination of a first portion of the opticalexcitation arrangement with an optical spectral filter, wherein saidcombination is configured to achieve at least 2.5 μm spatial resolutionin said image data.
 8. A method according to claim 7, wherein saiddirecting first light includes directing first light through a secondportion of the optical excitation arrangement that is different from thefirst portion.
 9. A method according to claim 7, comprising transmittingsaid fluorescence through an element of the optical excitationarrangement that is used in reflection during said delivering the firstlight.
 10. A method according to claim 5, further comprisingsequentially acquiring an optical signal from the pre-defined area atthe optical sensor array through a combination of a first portion of theoptical excitation arrangement with an optical spectral filter at afrequency rate sufficient to track cellular dynamics at said pre-definedarea.
 11. A method according to claim 5, wherein said receivingfluorescence includes interacting light originated at the pre-definedarea with first and second optical components each of which isconfigured as an optical spectral filter and a third optical componentconfigured as an achromatic lens.
 12. A method according to claim 5,further comprising removably positioning the optical sensor array ofsaid microscope system at a distance shorter than 1 inch from thepre-defined area to define a spatial resolution of the image dataacquired by the optical sensor array to be at least 2.5 μm while thefield-of-view of the optical excitation arrangement is at least 0.5 mm².13. A method according to claim 5, comprising propagating light towardsthe optical sensor array along a sinusoidal spatial path.
 14. A methodaccording to claim 5, wherein each of said delivering the first lightand said receiving fluorescence includes propagating light through amedium having radially variable refractive index.